CN116769717A - 一种靶向外泌体及其制备方法和应用 - Google Patents
一种靶向外泌体及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种靶向外泌体及其制备方法和应用,涉及生物医药技术领域。本发明首次将具有靶向肿瘤微环境的pHLIP与HEK393T来源的外泌体膜表面蛋白Lamp2b结合,制备得到具有靶向肿瘤作用的外泌体;并以该靶向外泌体作为载体来装载有抗肿瘤作用的中药单体莪术醇,成功构建具有靶向肿瘤作用的CUR‑ExosL2的载药系统,该载药系统在保持完整的外泌体结构、性质和功能的同时,实现了对莪术醇的高载药量。本发明通过基因工程对外泌体进行工程化改造实现药物靶向递送,实现胃腺肿瘤的特异性给药,改善药物的生物利用度,为胃癌的中药靶向治疗提供了新的思路和参考。
Description
技术领域
本发明涉及生物医药技术领域,具体涉及一种靶向外泌体及其制备方法和应用。
背景技术
胃癌(Gastric Cancer,GC)是全球常见的五大癌症之一,已被确定为全球癌症相关死亡的主要原因之一,根据国际癌症机构的统计,截止到2020年,全球胃癌新发病例100多万例,死亡病例约76.9万例。胃癌患者由于早期临床症状不显著,特异性指标较差,因此超过70%患者在确诊时处于晚期,错过手术和放疗的最佳时期。而传统的化疗药物和靶向治疗药物,由于耐药性和细胞毒性的副作用,使其在临床治疗效果逐渐不佳。因此,寻找新型抗癌药物具有重要意义,尤其是低毒、高效的抗肿瘤药物是当今世界研究的重点和热点。与合成的化学药物相比,来源于天然植物的中药有诸多优势,比如:低毒性、高安全性/途径多、靶点多等。
由于中药具有多种活性化合物的协同和药代动力学作用,使中药制剂在治疗各种疾病中发挥着突出的作用。目前临床上也有许多中药配方和中药单体已成功的用于抗肿瘤治疗。例如,中药单体-姜黄素,其抗肿瘤作用已被广泛研究,能够抑制肿瘤的发生、进展、侵袭和转移等。研究者发现,在中医药抗胃癌治疗中,橙皮素单体能够通过上调PTEN表达促进顺铂诱导的体外和体内胃癌细胞凋亡。Zhang等人发现木犀草素通过影响细胞周期进程抑制肿瘤细胞活性从而来诱导胃癌细胞SGC-7901的凋亡。尽管中药在临床研究中显示出良好的抗肿瘤性,但其往往存在稳定性低、水溶性差、体内代谢快、生物利用度低等缺点,使其在临床应用上受到限制,因此,急需一种稳定性和生物相容性良好的载体来改善其药物特性。
外泌体是由细胞内多泡体和细胞膜融合后,释放到细胞外的一种直径约30-150nm的膜性囊泡,其中包含RNA、蛋白质、DNA片段等多种胞内物质。外泌体能在细胞间穿梭,有利于细胞间物质与信息的交换,可装载化疗药物及siRNA进行靶向治疗。外泌体作为药物载体具有天然的优势:(1)具备纳米级尺寸,在体内很容易逃过网状内皮系统的捕获;(2)具有磷脂双分子层结构,与细胞膜组成相似,对细胞膜具有较强的亲和力,便于进入细胞;(3)属于内源性囊泡,作为载体进入体内不会引起免疫反应,不会被体内的免疫系统鉴定为“非我物质”而吞噬;(4)其来源于细胞,表面含有众多跨膜蛋白,可以通过基因修饰的方式,在适当的蛋白末端加上能够靶向目标细胞的配体短肽,实现体内的靶向治疗。
低pH插入肽(pH low insertion peptide,pHLIP)来源于细菌视紫红质的C-螺旋,是一种水溶性分子,以pH依赖性方式与脂质膜相互作用。pHLIP具有靶向酸性组织和双重递送能力,各种外源性分子可以通过与pHLIP的N端或者C端偶联,从而将它们靶向递送至细胞表面和细胞内。pHLIP已经被证明可以靶向肿瘤酸性微环境,其靶向递送系统对比传统的靶向纳米载体和穿膜肽具有特异性强的优势,在应用上pHLIP具有以下有利条件:pHLIP对肿瘤细胞中受体或抗原的异质性表达不敏感;pHLIP具有主动靶向酸性肿瘤微环境的特定标志;pHLIP不仅可以精准靶向原发肿瘤病灶和转移病灶,还可以利用pHLIP插入肿瘤细胞膜表面的特性开发其更多方面的应用。pHLIP技术可以递送不同类型的功能性分子到细胞中用于治疗和诊断,在临床具有较好的应用前景,但目前仍然有不足之处需要优化,其中提高pHLIP和外源性分子的合成效率可以显著提高其临床转化能力。虽然pHLIP已被证实对酸性肿瘤微环境有特异性靶向作用,但还需要进一步的提高其靶向的效率,在临床应用时需要筛查患者有无炎症性疾病,从而提高pHLIP的特异性。
发明内容
(一)解决的技术问题
针对现有技术的不足,本发明提供了一种靶向外泌体及其制备方法,所述外泌体用于制备具有靶向肿瘤作用的中药纳米载体和用于制备抗胃癌药物中的应用。
(二)技术方案
为实现以上目的,本发明通过以下技术方案予以实现:
一方面,本发明提供了一种靶向外泌体,所述外泌体是包含有低pH插入肽与外泌体表面的溶酶体相关膜蛋白Lamp2b形成的融合蛋白。
进一步地,所述外泌体是包含有Lamp2b信号肽-WT-pHLIP-Flag和Lamp2b-HA连接形成的pHILP-Lamp2b-1融合蛋白;其中所述融合蛋白的基因序列如SEQ ID NO.1所示;其中Lamp2b信号肽-WT-pHLIP-Flag序列如SEQ ID NO.2所示;Lamp2b-HA序列如SEQ ID NO.3所示。
进一步地,所述外泌体还包含有具有保护作用的糖基化基序的融合蛋白pHILP-Lamp2b-2,所述融合蛋白的基因序列如SEQ ID NO.4所示。
又一方面,本发明还提供了一种靶向外泌体的制备方法,所述外泌体按照以下步骤制备:
(1)利用重叠延伸PCR技术将合成的Lamp2b信号肽-pHLIP-Flag和Lamp2b-HA基因序列连接为融合蛋白pHLIP-Lamp2b-1和pHLIP-Lamp2b-2;
(2)将酸敏融合肽基因克隆至pCDH-CMV-MCS-EF1-GFP-Puro慢病毒载体上的多克隆位点,获得重组质粒pCDH-pHLIP-Lamp2b1-Puro和重组质粒pCDH-PHLIP-Lamp2b2-Puro,分别简称为L1和L2;
(3)将重组质粒pCDH-PHLIP-Lamp2b1-Puro和pCDH-PHLIP-Lamp2b2-Puro包装成慢病毒转染HEK293T细胞,并收集慢病毒液进行浓缩来感染HEK293T细胞;随后使用嘌呤霉素和倍数稀释法相结合来筛选单克隆细胞,获得稳定表达酸敏融合蛋白的HEK293T单克隆细胞株HEK293TL1/L2;
(4)外泌体的提取
将稳转单克隆HEK293T L1/L2细胞正常传代,待细胞融合度达70%-80%时,弃去培养瓶内培养基;随后加入PBS缓冲液洗涤细胞,洗涤后弃去PBS缓冲液;然后加入DMEM基础培养基进行饥饿培养,放入37℃,5%CO2细胞培养箱培养24-48h;收集细胞上清液,4℃,300×g离心10min,沉淀并去除活细胞;收集上清液,4℃,2000×g离心20min,沉淀并去除死细胞;收集上清液,4℃,10000×g离心30min,沉淀并去除死细胞碎片;收集上清液,并用0.22μm滤器过滤,去除大于220nm的囊泡,并收集滤液;将收集的滤液使用15mL超滤管进行超滤,截留分子量为100kDa;收集超滤液,4℃,100000×g离心70min,弃上清;加PBS缓冲液重悬沉淀,4℃,100000×g离70min,弃上清,沉淀即为外泌体。
又一方面,本发明还提供了一种靶向外泌体的应用,所述外泌体用于制备具有靶向肿瘤作用的中药纳米载体;所述中药单体为具有抗肿瘤作用的中药单体;所述中药单体包括但不限于莪术醇、姜黄素、双去甲氧基姜黄素、槲皮素、紫杉醇、白藜芦醇、雷公藤甲素、木犀草素、紫草素、金合欢素、常春藤皂苷、异鼠李素中的任意一种。
进一步地,所述靶向外泌体中药纳米载体采用室温共孵育的方法制备,具体按照以下步骤制备:
1)取4份纯化的外泌体(200μg/mL),分别与600、400、200、100μg/mL莪术醇溶液在室温下共孵育48h;
2)分别将外泌体和莪术醇混合液在4℃、120000×g离心70min,去除上清及游离的莪术醇,用10ml PBS洗涤重悬,再次4℃、120000×g离心70min,适量PBS重悬沉淀,即得到pHLIP-Lamp2b-1/2融合蛋白外泌体中药纳米载体。
另一方面,本发明提供的靶向外泌体的应用于制备治疗胃癌靶向药物。
(三)有益效果
本发明提供了一种靶向外泌体及其制备方法和应用。本发明首次将具有靶向肿瘤微环境的pHLIP与HEK393T来源的外泌体膜表面蛋白Lamp2b结合,制备得到具有靶向肿瘤作用的外泌体;并以该靶向外泌体作为载体来装载有抗肿瘤作用的中药单体莪术醇,成功构建具有靶向肿瘤作用的CUR-ExosL2的载药系统,该载药系统在保持完整的外泌体结构、性质和功能的同时,实现了对莪术醇的高载药量。本发明通过基因工程对外泌体进行工程化改造实现药物靶向递送,实现胃腺肿瘤的特异性给药,改善药物的生物利用度,为胃癌的中药靶向治疗提供了新的思路和参考。
本发明采用了差速离心法、超滤法和超高速离心相结合来提取HEK293T、L1和L2细胞来源的外泌体,先使用差速离心去除细胞杂质,再利用超滤管浓缩细胞上清液,富集外泌体,随后使用超高速离心提取外泌体。该方法明显提高外泌体的提取量和提纯度。
附图说明
图1为HEK293T细胞、HEK293TExo、L1-Exo和-L2-Exo标志蛋白表达情况。
图2为HEK293T-Exos(图2A)、L1-Exos(图2B)、L2-Exos(图2C)的粒径分布图。
图3为HEK293T-Exos(图3A)、L1-Exos(图3B)、L2-Exos(图3C)的透射电子显微镜图。
图4为Western Blot法检测HEK293T-Exos、L1-Exos、L2-Exos酸敏融合蛋白的表达情况。
图5为激光共聚焦下荧光染料pKH67标记HEK293T-Exos、L1-Exos、L2-Exos观察结果。
图6为激光共聚焦下观察胃癌细胞HGC-27对HEK293T-Exos的摄取情况。
图7为激光共聚焦下观察胃癌细胞HGC-27对L2-Exos的摄取情况。
图8为CUR-HEK293Texos(图8A)、CUR-L2Exo(图8B)透射电子显微镜图。
图9为CUR-L2Exo粒径分布图。
图10为莪术醇(S3)、CUR-L2Exos(S2)和CUR--HEK293TExos(S1)的HPLC图。
图11为莪术醇的标准曲线图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将结合本发明实施例,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
试验1
1、pHLIP-Lamp2b-1/2融合蛋白靶向外泌体的制备,按照以下步骤:
(1)Lamp2b-HA目的基因序列扩增
利用PCR扩增技术扩增出除信号肽以外的Lamp2b目的基因序列,并利用PCR技术在其序列末端添加HA标签序列,获得的Lamp2b-HA序列如SEQ ID NO.3所示;
(2)Lamp2b信号肽-WT-pHLIP-Flag基因序列扩增
利用PCR技术扩增Lamp2b信号肽-pHLIP-Flag,获得的Lamp2b信号肽-WT-pHLIP-Flag序列如SEQ ID NO.2所示;
(3)构建pHLIP-Lamp2b-1和pHLIP-Lamp2b-2融合蛋白基因
利用重叠延伸PCR技术将合成的Lamp2b信号肽-pHLIP-Flag和Lamp2b-HA基因序列连接为融合蛋白pHLIP-Lamp2b-1和pHLIP-Lamp2b-2;其中pHLIP-Lamp2b-2中含有保护作用的糖基化基序,所述糖基化保护位点的序列如SEQ ID NO.5所示;pHLIP-Lamp2b-1和pHLIP-Lamp2b-2的序列分别如SEQ ID NO.1和4所示。
(4)构建酸敏融合肽重组质粒
将酸敏融合肽基因克隆至pCDH-CMV-MCS-EF1-GFP-Puro慢病毒载体上的多克隆位点,获得重组质粒pCDH-pHLIP-Lamp2b1-Puro和重组质粒pCDH-pHLIP-Lamp2b2-Puro,分别简称为L1和L2;
(5)建立稳定表达酸敏融合蛋白的HEK293T单克隆细胞株HEK293TL1/L2
将重组质粒pCDH-pHLIP-Lamp2b1-Puro和pCDH-pHLIP-Lamp2b2-Puro包装成慢病毒转染HEK293T细胞,并收集慢病毒液进行浓缩来感染HEK293T细胞;随后使用嘌呤霉素和倍数稀释法相结合来筛选单克隆细胞,获得稳定表达酸敏肽融合蛋白的HEK293T单克隆细胞株HEK293TL1/L2;
(6)外泌体(Exos)的提取
将稳转单克隆HEK293T L1/L2细胞正常传代,待细胞融合度达70%-80%时,弃去培养瓶内培养基;随后加入PBS缓冲液洗涤细胞,洗涤后弃去PBS缓冲液;然后加入DMEM基础培养基进行饥饿培养,放入37℃,5%CO2细胞培养箱培养24-48h;收集细胞上清液,4℃,300×g离心10min,沉淀并去除活细胞;收集上清液,4℃,2000×g离心20min,沉淀并去除死细胞;收集上清液,4℃,10000×g离心30min,沉淀并去除死细胞碎片;收集上清液,并用0.22μm滤器过滤,去除大于220nm的囊泡,并收集滤液;将收集的滤液使用15mL超滤管进行超滤,截留分子量为100kDa;收集超滤液,4℃,100000×g离心70min,弃上清;加PBS缓冲液重悬沉淀,4℃,100000×g离心70min,弃上清,沉淀即为外泌体。
按照上述方法分别制备HEK293T、HEK293TL1、HEK293TL2细胞来源的外泌体,分别命名为HEK293T-Exos、L1-Exos、L2-Exos,并对所制备的外泌体进行鉴定。
2、pHLIP-Lamp2b-1/2融合蛋白靶向外泌体的鉴定
(1)Western blot检测外泌体特异性蛋白
Western Blot法检测Exos特异性标志蛋白的表达情况,其中包括膜蛋白CD81以及内含蛋白Alix、TSG101、HSP70。结果如图1所示,本实验从超离沉淀提取的蛋白,阳性表达CD81和Alix、TSG101、HSP70,并且表达量明显高于细胞,提示超高速离心法分离得到为Exos。
(2)动态光散射(DLS)测定粒径
DLS检测HEK293T-Exos、L1-Exos、L2-Exos的粒径,结果如图2所示,颗粒集中分布在159.2nm、159.5nm、151.5nm左右,且粒径分布呈单峰正态分布,多分散系数PDI为0.168、0.200、0.178左右。说明颗粒分散性较好,Exos的定义直径是30-150nm,该实验得到的HEK293T-Exos、L1-Exos、L2-Exos粒径符合Exos的范围。
(3)透射电子显微镜观察外泌体形态及大小
透射电子显微镜结果显示,超速离心所得HEK293T-Exos、L1-Exos、L2-Exos沉淀,直径范围30-150nm之间,具有明显的茶托样双层膜结构组成,呈单个分布,背景较为清晰,污染物少,如图3所示。
本发明采用了差速离心法、超滤法和超高速离心相结合来提取HEK293T、L1和L2细胞来源的外泌体,先使用超滤管浓缩细胞上清液,富集外泌体,再使用超高速离心提取外泌体。通过粒径分析。透射透射电子显微镜和Western blot方法鉴定超高速离心所获得沉淀为外泌体,可用于后续靶向性验证实验。
试验2
验证pHLIP-Lamp2b-1/2融合蛋白外泌体的靶向性
1、Western Blot检测外泌体标志蛋白
Western Blot法检测L1-Exos和L2-Exos酸敏融合蛋白的表达情况,L1-Exos和L2-Exos均表达酸敏融合蛋白。结果如图4所示,与L1-Exos对比,L2-Exos中pHLIP表达量更高,L1-Exos中pHLIP有明显被降解,结果表明在pHLIP前增加糖基化保护位点,可以有效防止其被降解。
2、染色标记外泌体
使用荧光染料pKH67标记HEK293T、HEK293TL1和HEK293TL2细胞来源的外泌体,并在激光共聚焦下观察外泌体的标记结果,结果如图5所示。
3、激光共聚焦观察胃癌细胞HGC-27对外泌体摄取情况
使用荧光染料pKH67标记HEK293T和HEK293TL2细胞来源的外泌体,与HGC-27细胞共孵育,为验证含有pHLIP-Lamp2b-2融合蛋白外泌体在不同酸碱环境下对肿瘤细胞的靶向性,分别将培养基调置不同的pH值,pH值分别设定为5.4、6.0、7.4。在与胃癌细胞HGC-27共孵育6h后,使用激光共聚焦显微镜来观察摄取的情况,结果如图6、图7所示。该结果表明在不同pH值条件下,胃癌细胞HGC-27对HEK293T-Exos和HEK293TL2-Exos摄取存在明显的差异性,其中在酸性环境下,HGC-27细胞对HEK293TL2-Exos摄取能力较强。结果证明HEK293TL2-Exos对胃癌细胞有靶向性。
试验3
1、pHLIP-Lamp2b-2融合蛋白外泌体中药纳米载体的制备
1)取4份纯化的外泌体(200μg/mL),分别与600、400、200、100μg/mL莪术醇溶液在室温下共孵育48h;
2)分别将外泌体和莪术醇(CUR)混合液在4℃、120000×g离心70min,去除上清及游离的莪术醇,用10ml PBS洗涤重悬,再次4℃、120000×g离心70min,适量PBS重悬沉淀,即得到pHLIP-Lamp2b-2融合蛋白外泌体中药纳米载体,简称为CUR-L2Exos。
按照上述方法同时制备CUR-HEK293TExos。
2、CUR-L2Exos的表征
(1)透射电子显微镜观察CUR-L2Exos形态及大小
透射电子显微镜结果显示,载药后的CUR-HEK293TExos、CUR-L2Exo与载药前Exos相比未出现较大变化,依然具有明显的茶托样双层膜结构,超声孵育后膜恢复较好,说明该载药方法对外泌体形态无明显影响,结果如图8所示。
(2)动态光散射(DLS)测定CUR-L2Exos粒径
DLS检测CUR-L2Exos的粒径,结果如图9所示,颗粒集中分布在170nm左右,且粒径分布呈单峰正态分布,多分散性指数(PDI)分别为0.149。说明颗粒分散性较好,与载药前外泌体相比,载药后的外泌体的粒径略有增大。
3、利用高相液相色谱对CUR-L2Exos中莪术醇的含量测定
(1)CUR-L2Exos的专属性
莪术醇和CUR-Exos的色谱峰保留时间为16.9min,峰形较好,Exos在该时间点无色谱峰信号,说明外泌体对莪术醇的测定无干扰,专属性良好。结果如图10所示。
(2)莪术醇的标准曲线
分别配制浓度为8、16、32、64、128和256μg/mL的莪术醇对照品溶液,按“色谱柱:YMC-triart C18柱(250mm×4.6mm,5μm);流动相:乙腈-磷酸水(60∶40,v/v);流速:1mL/min;柱温:30.0℃;进样量:10μL;检测波长:200nm”色谱条件进样测定,以绘制标准曲线。
莪术醇标准曲线图如图11所示,线性回归方程为Y=23.27x+8.246(R2=0.9999),莪术醇在8-256μg/mL的质量浓度范围内线性关系良好。
(3)CUR-L2Exos载药量的测定
分别取CUR-L2Exos破乳溶液100μL,按“色谱柱:YMC-triart C18柱(250mm×4.6mm,5μm);流动相:乙腈-磷酸水(60∶40,v/v);流速:1mL/min;柱温:30.0℃;进样量:10μL;检测波长:200nm”色谱条件进样测定莪术醇的含量,计算载药量(LC)。LC(%)=W1/W2×100%,其中,W1为包封的莪术醇总量;W2为CUR-L2Exos的蛋白含量。
通过莪术醇的标准曲线回归方程,计算得到CUR-L2Exos溶液中莪术醇的含量为:77.84ug,即CUR-L2Exos的载药率为19.46%。
以上实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
Claims (7)
1.一种靶向外泌体,其特征在于,所述外泌体包含有低pH插入肽与外泌体表面的溶酶体相关膜蛋白Lamp2b形成的融合蛋白。
2.根据权利要求1所述的一种靶向外泌体,其特征在于,所述外泌体是包含有Lamp2b信号肽-WT-pHLIP-Flag和Lamp2b-HA连接形成的pHILP-Lamp2b-1融合蛋白;其中所述融合蛋白的基因序列如SEQ ID NO.1所示;其中Lamp2b信号肽-WT-pHLIP-Flag序列如SEQ ID NO.2所示;Lamp2b-HA序列如SEQ ID NO.3所示。
3.根据权利要求1所述的一种靶向外泌体,其特征在于,所述外泌体还包含有具有保护作用的糖基化基序的融合蛋白pHILP-Lamp2b-2,所述融合蛋白的基因序列如SEQ ID NO.4所示。
4.一种靶向外泌体的制备方法,其特征在于,所述外泌体按照以下步骤制备:
(1)利用重叠延伸 PCR 技术将合成的Lamp2b信号肽-pHLIP-Flag和Lamp2b-HA 基因序列连接为融合蛋白pHLIP-Lamp2b-1和 pHLIP-Lamp2b-2;
(2)将酸敏融合肽基因克隆至 pCDH-CMV-MCS-EF1-GFP-Puro 慢病毒载体上的多克隆位点,获得重组质粒 pCDH-pHLIP-Lamp2b1-Puro和重组质粒 pCDH-pHLIP-Lamp2b2-Puro,分别简称为L1和L2;
(3)将重组质粒pCDH-pHLIP-Lamp2b1-Puro和 pCDH-pHLIP-Lamp2b2-Puro 包装成慢病毒转染 HEK293T 细胞,并收集慢病毒液进行浓缩来感染 HEK293T 细胞;随后使用嘌呤霉素和倍数稀释法相结合来筛选单克隆细胞,获得稳定表达酸敏融合蛋白的HEK293T 单克隆细胞株 HEK293TL1、HEK293TL2;
(4)外泌体的提取
将稳转单克隆HEK293TL1、HEK293TL2细胞正常传代,待细胞融合度达 70%-80%时,弃去培养瓶内培养基;随后加入 PBS 缓冲液洗涤细胞,洗涤后弃去 PBS 缓冲液;然后加入DMEM 基础培养基进行饥饿培养,放入 37℃,5%CO2 细胞培养箱培养 24-48h;收集细胞上清液,4℃,300×g离心10min,沉淀并去除活细胞;收集上清液,4℃,2000×g 离心20min,沉淀并去除死细胞;收集上清液, 4℃,10000×g 离心30min,沉淀并去除死细胞碎片;收集上清液,并用 0.22μm滤器过滤,去除大于 220nm 的囊泡,并收集滤液;将收集的滤液使用15mL 超滤管进行超滤,截留分子量为 100kDa;收集超滤液,4℃,100000×g 离心 70min,弃上清;加 PBS 缓冲液重悬沉淀,4℃,100000×g 离心70min,弃上清,沉淀即为外泌体。
5.一种靶向外泌体的应用,其特征在于,所述外泌体用于制备具有靶向肿瘤作用的中药纳米载体;所述中药单体为具有抗肿瘤作用的中药单体;所述中药单体包括但不限于莪术醇、姜黄素、双去甲氧基姜黄素、槲皮素、紫杉醇、白藜芦醇、雷公藤甲素、木犀草素、紫草素、金合欢素、常春藤皂苷、异鼠李素中的任意一种。
6.根据权利要求3所述的一种靶向外泌体的应用,其特征在于,靶向外泌体中药纳米载体采用室温共孵育的方法制备,具体按照以下步骤制备:
1)取4份浓度为200μg/mL纯化的外泌体,分别与600、400、200、100 μg/mL 莪术醇溶液在室温下共孵育 48h;
2) 分别将外泌体和莪术醇混合液在4℃、120000×g离心70 min,去除上清及游离的莪术醇,用 10 ml PBS洗涤重悬,再次4℃、120000×g 离心70min,适量PBS 重悬沉淀,即得到靶向外泌体中药纳米载体。
7.一种靶向外泌体的应用,其特征在于,所述外泌体用于制备治疗胃癌靶向药物。
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